Production of reprogrammed cells with restored potential

ABSTRACT

A method for treating cells and/or nuclear transfer units and/or stem cells in culture with such compounds, individually or in combinations, is described. The method results in a globally hypomethylated genome and a restoration of cell differentiation and/or developmental potential, or potentiality. In addition, a method for the in vitro production of reprogrammed cells which have had differentiation potential (totipotential, pluripotential, or multipotential) restored by demethylating the genome is described.

CROSS-REFERENCE TO RELATED APPLICATIONS

Priority is hereby claimed to provisional application Ser. No.60/704,465, filed Aug. 1, 2005, which is incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates generally to the fields of cell biology, stemcells, cell differentiation, somatic cell nuclear transfer andcell-based therapeutics. More specifically, this invention is directedto methods and products for cell reprogramming and cell-basedtherapeutics. Methods of isolation and culture, as well as therapeutic'uses for the isolated cells are also provided.

DESCRIPTION OF THE PRIOR ART

Recent dietary studies have described an interaction between methyldonor status, single-carbon metabolism and DNA methylation [1-12]. DNAmethylation is associated with developmental status and differentiationpotential [13-19]. Moreover, a clear requirement for somatic cellnuclear transfer (SCNT)-induced reprogramming and reprogrammingassociated with normal fertilization is DNA demethylation [20]. It iswithin this context that the following background material is reviewed.

During development of multicellular organisms, different cells andtissues acquire different programs of gene expression. These distinctgene expression patterns appear to be substantially regulated byepigenetic modifications such as DNA methylation, histone modificationsand various chromatin-binding proteins [21, 22]. Thus each cell typewithin a multicellular organism has a unique epigenetic signature whichis thought to become “fixed” once cells differentiate or exit the cellcycle.

However, some cells undergo major epigenetic “reprogramming” duringnormal development or certain disease situations. Reprogramming involvesthe removal of epigenetic marks in the nucleus, followed byestablishment of a different set of marks [23-25]. For example, atfertilization, gametic epigenetic marks are erased and replaced withembryonic marks required for totipotency and embryonic development.Reprogramming also occurs in primordial germ cells where parentalimprints are erased to restore totipotency. Cancer cells and cells thattransdifferentiate also are thought to undergo reprogramming. Finally,dramatic reprogramming is required following SCNT for the purposes ofreproductive cloning and stem cell therapy [26-33].

Recently, new insights have been obtained into epigenetic reprogrammingin normal development and SCNT. Although insights into the mechanisms ofreprogramming and the factors involved are still rudimentary, thefoundational knowledge is now reaching a stage at which more detailedconcepts can be developed and novel experimental approaches can bedevised to examine mechanistic aspects that will provide novelcommercial opportunities. A brief summary of in vivo-induced(fertilization and embryogenesis) and in vitro-induced SCNTreprogramming follows.

Epigenetic Reprogramming and Development In VivoReprogramming—Fertilization

DNA Methylation: In mice, at fertilization, the parental genomes are indifferent stages of the cell cycle with distinct epigenetic marks andchromatin organization. The paternal genome delivered by the maturesperm is single copy and is packaged densely with protamines. Thematernal genome is arrested at metaphase II and its two-copy genome ispackaged with histones. At fertilization, the protamines are replacedwith histones and the maternal genome completes meiosis. After histoneacquisition, the paternal genome undergoes a genome-wide loss of DNAmethylation as detected by indirect immunofluorescence [34] andbisulphite sequencing [35]. Demethylation is completed before DNAreplication begins in the paternal pronucleus. It should be noted,however, that not all regions of the genome are demethylated at thisstage. It is believed that the oocyte cytoplasm contains demethylationfactors that specifically target or exclude certain sequence classes insperm chromatin.

Histone Modification: Maternal chromatin is organized so that DNAmethylation and chromatin modifications are abundant at fertilization.These include both nucleohistone modifications and chromatin proteinsassociated with active and repressive states. For example, histonemodifications typically associated with an active state, such asacetylated lysine and H3K4me are found in the female pronucleus [34, 36,37]. Heterochromatic modifications such as H3K9me2/3, H3K27mel andH41C20me3, largely associated with a repressive chromatin state, arealso present.

Histones associated with the male pronucleus are highly acetylated [34,36]; however, immediately upon histone incorporation, H3K4mel, H3K9meland H3K27mel are detectable [37-39]. This occurs when DNA methylation isstill present in the male pronucleus. The progressive histonemodifications occurring to the paternal genome presumably lead to achromatin state equivalent to that of the maternal genome.

Preimplantation Development

Passive demethylation: From the one cell to blastocyst stage in themouse, there are further changes in global DNA methylation. DNAmethylation is reduced progressively with each cleavage division andthis loss is dependent on DNA replication [40, 41]. Dnmt1o, a DNMT1protein inherited from the oocyte, is excluded during the first threecleavage divisions [42, 43], accounting for the loss of methylation by apassive mechanism.

Histone modification: The degree to which histone modifications arereprogrammed during passive DNA demethylation is unclear at present.

Epigenetic Asymmetry and Lineage Commitment

Methylation: The first lineage allocation event in mammalianembryogenesis occurs at the morula stage and results in the formation ofthe Inner Cell Mass (ICM) and trophoectoderm (TE) lineages in theblastocyst. The mechanism for the cell fate decision is unknown.Interestingly, two-cell blastomeres are still totipotent but by thefour-cell stage (in mice) lineage bias is present based on decreasedtotipotential [44]. Global differences in DNA methylation betweenextraembryonic and embryonic lineages have been detected and thedifferences are detectable as early as the blastocyst stage at whichcombinations of active and passive demethylation have resulted in alower state of methylation in the TE. In contrast, the ICM has anincreased level of global methylation due to de novo methylation likelycaused by DNMT3b, a de novo DNA methyltransferase, which is detectablein blastocyst ICM, but not TE [34].

Histone modification: In mice, histone H3K9me3 marks heterochromaticfoci in the ICM and H3K27mel, met and me3 are more abundant in the ICMthan the TE [38]. The inactive X chromosome and certain imprintedregions are marked in the TE by H327me and H3K9me. In general, as withDNA methylation, higher levels of specific repressive histonemethylation marks also are found in the ICM when compared to TE. Thusepigenetic asymmetry is required for development.

Somatic Cell Nuclear Transfer: One method of altering epigenetic marksexperimentally is by SCNT or cloning. SCNT requires that adifferentiated somatic cell nucleus be reprogrammed to a state oftotipotency in an oocyte environment in which nuclear DNA material hasbeen removed (often referred to as “enucleation”). All epigenetic marksthat have been examined in cloned embryos and adults from multiplespecies show abnormalities and most SCNT embryos also differ from eachother in the precise epigenetic profile they possess, indicating thatSCNT-induced epigenetic reprogramming is a haphazard and stochasticprocess [30-33, 45].

In addition, development of SCNT embryos is highly variable with thevast majority dying at all stages of development, and the survivorshaving a variety of abnormalities. Of those that develop to latergestation stages or to term, many have placental abnormalities and asignificant proportion die perinatally from poor adaptation toextrauterine life. Because offspring of cloned animals appear normal,most developmental problems of clones likely are caused by epigeneticdefects. Both the development and the epigenetic abnormalities of SCNTembryos tend to be more severe the earlier they are examined, with lessabnormal ones surviving to later stages [46].

Epigenetic defects described in cloned offspring and SCNT embryosinclude errors in X-inactivation [47, 48], imprinting [49-51], DNAmethylation (both global and specific loci) [36, 47, 52, 53], histoneacetylation [43], methylation [43] and alterations in gene expression[54] including the failure to reactivate Oct4, a key pluripotency anddevelopmentally important gene [55-57].

Reprogramming induced by SCNT therefore appears to have at least twoimportant requirements: removal of nuclear proteins and demethylation ofsomatic cell DNA, the latter apparently being the most important. Forexample, Somonsson & Gurdon observed considerable delay of Oct4expression when methylated somatic nuclei from mouse thymus cells wereinjected into Xenopus oocytes [20]. The delay declined when the nucleiwere deproteinized prior to nuclear transfer. However when unmethylatedDNA, e.g., bacterial plasmid, was injected, there was no detectabledelay. This indicates that when all repressive proteins have beenremoved from nuclei, a substantial delay still occurs and that DNA mustbe demethylated before transcription can ensue. In addition, using ahypomorphic allele of Dnmt1, Blelloch et al demonstrated demethylationof somatic donor cells improved cloning efficiency and production ofembryonic stem cells in vitro indicating restoration of differentiationand developmental potential of a differentiated cell is enabled byreduced DNA methylation [136].

Mechanisms for Removal of Methylation from DNA Not Understood: Theremoval of methylation from DNA (or histones for that matter) is notunderstood mechanistically. The loss of DNA methylation of the paternalgenome in the zygote is likely to be an enzyme-catalyzed, activedemethylation. An oocyte also can actively demethylate a transferredsomatic nucleus, suggesting the activity is present. Several candidatebiochemical pathways have been suggested that would either remove themethyl group in the C5 position of the cytidine ring directly (directdemethylation) or the cytidine base (indirect demethylation). To date,direct methods to remove the methyl group have either not been describedor have not been validated.

Indirect pathways to demethylation involve DNA repair: For example, DNAglycosylases such as thymidine DNA glycosylase and methyl-binding domainprotein 4 normally repair T:G mismatches thought to result fromspontaneous deamination of 5 meC. More recently it has been shown thatactivation-induced cytidine deaminase and Apobec1, cytidine deaminases,can deaminate 5 meC to result in T and that these enzymes are expressedin oocytes and germ cells. This activity of cytidine deaminases coupledwith base excision repair theoretically could result in demethylationwithout DNA replication, but would still require extensive base excisionrepair in the early zygote.

Environmental Factors: A growing body of evidence suggests modifiableenvironmental factors such as diet can influence DNA methylation. Forexample, prenatal feeding of a methyl-supplemented diet can increase thelevel of DNA methylation and phenotypic expression of genes inoffspring. The coat color in mice is determined by agouti geneexpression. This is determined by the DNA methylation status of the longterminal repeat of the agouti gene in the hair follicle. If this regionis hypermethylated, the mouse is agouti in color, whereas if the mouseis hypomethylated the mouse is yellow. When pregnant female mice werefed a methyl-supplemented diet enriched in zinc, methionine, betaine,choline, folate, and vitamin B₁₂, there was an alteration in themethylation status of the agouti long terminal repeat and none of thepups had a yellow coat [58]. Thus, in utero exposure to nutrients canlead to epigenetic modifications of the genome in the offspring.

Folate's Role in DNA Methylation: Folate, a water soluble B-vitamin,plays a significant and modulatory role in DNA methylation status. Thesole biochemical function of folate is mediating the transfer ofone-carbon moieties [59-61] and thus has a central role in one-carbonmetabolism. Animal studies have further shown that folate deficiencycauses genomic and gene specific hypomethylation in rat liver and thedegree appears to depend on the severity and duration of folatedepletion [62, 63]. DNA hypomethylation also has been identified inlymphocytes of humans on low dietary folate and can be reversed byfolate repletion [64]. The effect of folate deficiency with respect toDNA methylation, however, is also highly complex. For example, folatedeficiency with or without reductions in DNMT1, did not affect overallgenomic DNA methylation levels or the methylation levels of twocandidate genes E-cadherin and p53, in normal or neoplastic tissue.

One-Carbon Metabolism

The Importance of SAM:SAH Ratios and Nutrient Role in Regulating DNAMethylation: The availability of nutrients can play an important role inregulating DNA methylation. Moreover, factors involved in one-carbonmetabolism also likely play an important role in methylation statusbecause they influence the supply of methyl groups and therefore thebiochemical pathways of methylation processes. Methyl groups suppliedfrom the diet, in the form of choline and methionine, or from thefolate-dependent one-carbon pool, must be activated toS-adenosylmethionine (SAM) to serve as substrates in transmethylationreactions. Because S-adenosylhomocysteine (SAH) is a product oftransmethylation reactions and a potent inhibitor of SAM-dependentmethyltransferases, the ratio of SAM:SAH is an important index oftransmethylation potential [65, 66]. When the ratio is high, methylationpotential is high. When the ratio is low, methylation potential is low.It is well known that folate depletion alone is a sufficient perturbingforce to diminish SAM pools. This leads to an increase in cellularlevels of SAH because the equilibrium of the SAH-homocysteineinterconversion actually favors SAH synthesis. Therefore whenhomocysteine metabolism is inhibited, as in folate deficiency, cellularSAH levels will be increased. Increased SAH inhibits methyltransferasesactivity and consequently DNA methylation reactions.

Glycine-N-methyltransferase (GNMT) Optimizes Transmethylation Reactions:The cytosolic enzyme GNMT functions to optimize transmethylationreactions by regulating the SAM:SAH ratio. When methyl groups areabundant and SAM levels are elevated, GNMT disposes of the excess methylgroups by forming sarcosine from glycine. SAM also reduces the supply ofmethyl groups originating from the one-carbon pool by inhibiting5,10-methylenetetrahydrofolate reductase (MTHFR) [67, 68], the enzymeresponsible for the synthesis of 5-methyletrahydrofolate (5-methyl-THF).5-Methyl-THF is folate coenzyme that donates its methyl group tohomocysteine to form methionine. Because 5-methyl-THF also binds to GNMTand inhibits its activity [69, 70], a decrease in 5-methyl-THF levelsdue to inhibition of MTHFR by SAM results in an increase in the activityof GNMT. Factors that activate GNMT lead to downregulation ofmethyltransferases, including DNA methyltransferases.

To date, there have been no reports testing the hypothesis thatactivation of GNMT in cells in culture results in reduced DNAmethylation. However, a few reports have described dietary manipulationof GNMT in rodents [71-73]. For example, diets supplemented with vitaminA and vitamin A derivatives, e.g. all-trans retinoic acid (ATRA), resultin increased GNMT activity and hypomethylated DNA in rat hepatocytes,while decreases in dietary folate, choline, betaine and vitaminsB_(6 & 12) also result in decreased DNA methylation in specific tissues.Thus, manipulations of methionine cycle components and adjusting thenutrient availability of methyl donors represent possible in vitroapproaches to demethylate DNA and restore potential. To date, no priorattempts to reprogram or alter the efficiency of reprogramming bymodifying components involved in single-carbon metabolism, alteringnutrient availability, and/or altering GNMT have been reported.

Current Methods for Inducing Cell Reprogramming: Current methods forinducing cell reprogramming in mammals depend primarily upon SCNT,involving nuclear reprogramming and to a lesser extent exposure to cellextracts, fusion of cell types and removal of nuclear proteins. None ofthese methods, however, overcome the primary rate-limiting step ofreducing DNA methylation levels required for efficient reprogramming.

Nuclear Reprogramming Inefficient: Nuclear reprogramming is dependent onchromatin state and specifically demethylation of DNA, which istypically inefficient or slow. As an example, SCNT cloning requiresefficient reprogramming of gene expression to silence donor cell geneexpression and activate an embryonic pattern of gene expression. Recentobservations indicate that reprogramming may be initiated by earlyevents that occur soon after SCNT, but then continues as developmentprogresses through cleavage and probably gastrulation. Becausereprogramming is slow and progressive, NT units have dramaticallyaltered characteristics as compared to fertilized embryos.

The methods described above for nuclear transfer are generally known tothe industry, and have been or are being applied to applications fordirecting differentiation of stem cells. However, nearly all of theapplications depend upon nuclear transfer technologies. Geron (MenloPark Calif.) has used embryonic stem cells to produce nerve cells andheart muscle cells and has made advances in manipulating stem cells toproduce other kinds of tissue. Infigen, Inc. (DeForest, Wis.) alsoreceived several nuclear transfer patents primarily directed tolivestock cloning applications.

In other attempts to improve efficiency of nuclear transfer, Dr. RudolphJaenisch of MIT, Whitehead Institute uses cDNA microarrays to establishthe molecular criteria for nuclear reprogramming specifically in nucleartransfer. Dr. Jaenisch's primary focus is on human disease such ascancer, and reprogramming cancers, and designing strategies to improveepigenetic reprogramming. H is main efforts have been focused onunderstanding the mechanisms that establish DNA methylation andreprogramming the methylated genome. Dr. Jaenisch is attempting todevelop a nuclear transfer-generated unit devoid of methylation byknocking out DNMT1 in donor cell lines and using the lines to produce NTunits [136].

Dr. Keith Latham of Temple University is examining how inefficientreprogramming affects cloned embryos. Dr. Latham's research focusesprimarily on physiologic data—examining changes in phenotypes aftercloning [137, 139].

Dr. Kevin Eggan, of Harvard University is assessing how thedevelopmental fate of an adult cell can be restored (reprogrammed),using nuclear transfer as a model, and made headlines in 2005 by fusinga human skin cell with an embryonic stem cell to create a hybrid thatlooked and acted like the stem cell, potentially laying a foundation forcreating tailor-made, genetically matched stem cells for patients [138].

Among other methods under investigation for production of stem cells orstem-like cells are the practices of Uri Verlinsky, of the ReproductiveGenetics Institute (In Vitro Fertilization Clinic, Chicago, Ill.).Verlinsky employs a method that uses a cell fusion process for fusingsomatic cells with stem cells, on the theory that the molecules of thesomatic cells will direct the differentiation of the stem cells into thesame type of cell as the somatic cell. Verlinsky has reportedly produced10 embryonic stem cell lines using his new StemBrid (stem/hybrid)technique. No embryos are used.

SUMMARY OF THE INVENTION

An object of the present invention is directed to a process forrestoring cell differentiation potential by reducing global DNAmethylation of the cell.

A further object of the present invention is directed to the treatmentof cells and/or nuclear transfer (NT) units and/or stem cells in cultureto yield a globally hypomethylated genome and restored potential of thecell for differentiation (e.g., pluripotential, multipotential, and/ortotipotential), defined as the cell's ability to differentiate into newcell types.

The present invention is directed to a method for reprogramming aeukaryotic cell. The method comprises, as step (a), decreasingS-adenosylmethione-to-S-adenosylhomocysteine ratio (SAM-to-SAH ratio) inthe eukaryotic cell. The SAM-to-SAH ratio can be decreased to 0.1 orless, preferably 0.5 or less, and most preferably 1.00 or less. Step (a)can include contacting the cell with a siRNA selected from the groupconsisting of SEQ. ID. NOS: 1-41. Further, step (a) can includeincreasing expression, activity, or both expression and activity ofglycine-N-methyl transferase within the eukaryotic cell. Step (a) canalso include contacting the cell with an amount of a retinoic acideffective to increase expression, activity, or both expression andactivity of glycine-N-methyl transferase within the cell. In addition,the method includes, as step (b), reducing levels of 5-methylcytosine inDNA within the eukaryotic cell. It is within the scope of the presentinvention to perform steps (a) and (b) simultaneously. Step (b) alsoincludes specifically suppressing expression, activity, or expressionand activity of DNA methyltransferases within the eukaryotic cell. Step(b) can also comprise contacting the cell with a DNA methyltransferaseinhibitor. Further, step (b) can comprise contacting the cell with asuppression-effective amount of a small-interfering ribonucleic acid(siRNA) dimensioned and configured to suppress expression of DNAmethyltransferases.

The present invention is also directed to a method for reprogramming aeukaryotic cell. The method comprises, as step (a), specificallysuppressing expression, activity, or expression and activity of DNAmethyltransferases within a eukaryotic cell; and simultaneously, as step(b), increasing expression, activity, or both expression and activity ofglycine-N-methyl transferase within the eukaryotic cell. Step (a) cancomprise contacting the cell with a suppression-effective amount of asiRNA dimensioned and configured to suppress expression of DNAmethyltransferases. Step (a) can also comprise contacting the cell witha suppression-effective amount of a siRNA dimensioned and configured tosuppress to reduce 5-methylated cytosines in DNA in the cell by at leastabout 5%. Step (b) can comprise contacting the cell with an amount of aretinoic acid effective to increase expression, activity, or bothexpression and activity of glycine-N-methyl transferase within the cell.Step (b) can also comprise contacting the cell with all-trans retinoicacid. Further, step (b) can include contacting the cell with a compoundthat binds a retinoic acid receptor. In addition to steps (a) and (b),the method can includes, as step (c), specifically suppressing withinthe cell expression, activity, or expression and activity of an enzymeselected from the group consisting of 5, 10-methylenetetrahydrofolatereductase and cystathione-beta-synthase. It is within the scope of thepresent invention to contact the cell with a siRNA selected from thegroup consisting of SEQ. ID. NOS: 1-41.

The present invention is further directed to a method to changedifferentiation potential in a eukaryotic cell. Here, the methodcomprises contacting the eukaryotic cell in vitro with an amount of asiRNA, wherein the siRNA is dimensioned and configured to suppressexpression of DNA methyltransferases specifically, provided that thesiRNA does not suppress expression of glycine-N-methyl transferase, andwherein the amount is effective to induce genome-wide DNA demethylation,wherein cellular differentiation potential is increased. The eukaryoticcells are selected from the group consisting of stem cells, progenitorcells, somatic cells, and cells subjected to somatic cell nucleartransfer (NT cells). This methods comprises contacting the cell with asiRNA that is dimensioned and configured to suppress specifically theexpression of a DNA methyltransferase selected from the group consistingof DNA methyltransferase 1 (Dnmt 1), DNA methyltransferase 3a (Dnmt 3a),and DNA methyltransferase 3b (Dnmt 3b). The method further comprisescontacting the cell with an amount of a compound effective to increaseexpression of, activity of, or both expression of and activity of,glycine-N-methyl transferase, such as retinoic acid or all-transretinoic acid. The cells can also be contacted with a compositioncomprising, in combination, the siRNA and all-trans retinoic acid.

The present invention is further directed to a method to increasedifferentiation potential in a differentiated cell, the methodcomprising contacting a differentiated cell, in vitro, with acomposition comprising, in combination, as step (i) aknockdown-effective amount of a small-interfering ribonucleic acid(siRNA), wherein the siRNA is dimensioned and configured to suppressexpression of DNA methyltransferases specifically; (ii) aknockdown-effective amount of a siRNA dimensioned and configured tosuppress expression of an enzyme selected from the group consisting of5, 10-methylenetetrahydrofolate reductase and cystathione-beta-synthase;and (iii) a compound effective to increase expression of, activity of,or both expression of and activity of, glycine-N-methyl transferase,wherein the cell is contacted with the composition for a time effectiveto induce genome-wide DNA demethylation within the cell, wherebycellular differentiation potential is increased. The method comprisescontacting the cell with a composition comprising a siRNA that isdimensioned and configured to suppress specifically the expression of aDNA methyltransferase selected from the group consisting of DNAmethyltransferase 1 (Dnmt 1), DNA methyltransferase 3a (Dnmt 3a), andDNA methyltransferase 3b (Dnmt 3b). The compound effective to increaseexpression of, activity of, or both expression of and activity ofglycine-N-methyl transferase, is retinoic acid or all-trans retinoicacid. The cells are contacted with a siRNA selected from the groupconsisting of SEQ. ID. NOS: 1-41.

The present invention is further directed to a method for determiningthe developmental potential of a cell. The method comprises determiningSAM-to-SAH ratio in the cell, wherein a SAM-to-SAH ratio of less thanabout 1.0 indicates increased developmental potential of the cell. Themethod comprises measuring methylation of cytosine residues present inthe DNA of the cell, wherein methylation less than about 10% indicatesincreased developmental potential of the cell.

The present invention further comprises a method for controllingdifferentiation potential in a cell, the method comprising, as step (a)maintaining SAM-to-SAH ratio in the cell to less than about 1.0; and (b)maintaining methylation of cytosine residues in DNA of the cell to lessthan about 40%. The method further comprises, as step (a) maintainingthe SAM-to-SAH ratio in the cell to less than about 0.5; and, as step(b) maintaining the methylation of cytosine residues in DNA of the cellto less than about 20%.

The present invention is further directed to a cell culture medium forincreasing differentiation potential in a differentiated cell disposedwithin the culture medium. The culture medium comprises a base mediumsufficient to maintain viability of the cell, in combination with afactor that reduces activity of methyl donors present in the cellculture medium. The factor is selected from the group consisting ofhomocysteine and all-trans retinoic acid. Alternatively, the factor is asuppression-effective amount of a siRNA dimensioned and configured tosuppress expression of DNA methyltransferases.

The present invention is also directed to a method for identifyingcompounds that affect cell differentiation. The method comprises, asstep (a), reducing percentage of cytosines in DNA of a cell that aremethylated by at least about 5%, wherein the cell has a first phenotypeprior to the reduction; and, as step (b), reducing SAM-to-SAH ratio inthe cell to less than about 1.0; and, as step (c) contacting the cellwith a compound suspected of affecting cell differentiation; and, asstep (d) comparing the first phenotype of the cell to phenotype of thecell after step (c), wherein appearance after step (c) of a phenotypedifferent from the first phenotype indicates that the compound has anaffect on cell differentiation. Step (a) can include reducing thepercentage of cytosines in the DNA of the cell that are methylated by atleast about 5%. Step (a) can also include reducing the percentage ofcytosines in the DNA of the cell that are methylated by at least about10%. The cell can include a reporter system to monitor changes intranscription, which measures presence of an enzyme activity selectedfrom the group consisting of luciferase activity, beta-lactamaseactivity, and beta-galactosidase activity

The present invention is further directed to a method of restoring celldifferentiation potential comprising:

(a) reducing DNA methylation in somatic cell nuclei, cells, and/orspecific parts of cells by reducing methyl donors in vitro;

(b) increasing expression of GNMT gene and/or enzyme activity thatresult in decreased SAM:SAH ratio and decreased levels of DNAmethylation;

(c) reducing SAM:SAH ratio by reducing expression and/or activity ofspecific enzymes such as MTHFR and/or cystathione-beta-synthase (Cbs);

(d) reducing the activity of DNA methyltransferases including Dnmts 1,3a and/or 3b;

(e) increasing homocysteine and/or SAH levels in vitro, glucagons, orrelated factors to lower the SAM:SAH ratio (reduced SAM: SAH will lowermethylation);

(f) depleting glutathione levels to decrease SAM:SAH ratio;

(g) developing defined media that incorporate one or more of the abovefactors to reduce methylation (medias deficient in methyl donors, mediaswith additional homocysteine, etc.);

(h) developing a new assay based on using a beta-lactamase reporter geneand/or other viable reporter genes (luciferase, beta glucosidase, orothers) to identify factors that induce transcription of the GNMT gene,or that identify other factors or activities that stimulate GNMTactivity; and

(i) developing a new assay based on using a beta-lactamase reporter geneand/or other viable reporter genes (luciferase, beta glucosidase, orothers) to identify factors that reduce transcription of the DNAmethyltransferase genes, or that identify other factors or activitiesthat reduce the activity of DNA methyltransferases.

The invention includes but is not limited to:

(a) new research tools including specialized media and cell culture thatenable in vitro production of reprogrammed cells with restoreddifferentiation potential (totipotential, pluripotential,multipotential) by demethylating the genome;

(b) new stem-like cells produced by these media and cultures, that canbe used for research or therapeutic applications;

(c) a new assay system for monitoring GNMT expression using a betalactamase reporter gene, luciferase, beta galactosidase, and/or otherrelevant reporter genes, to assess the impact of GNMT expression orother activity on restoring differentiation potential of the cell;

(d) a new assay system for monitoring DNA methyltransferase expressionusing a beta lactamase reporter gene, luciferase, beta galactosidase,and/or other relevant reporter genes, to assess the impact of DNAmethyltransferase expression or other activity on restoringdifferentiation potential of the cell;

(e) improving the efficiency of somatic cell nuclear transfer; and

(f) improving the derivation of embryonic stem cells from SCNTreconstructed embryos (or units).

The embodiments include:

(a) treating existing commercial or other media with ATRA, whichincreases GNMT transcription and activity, lowers the SAM:SAH ratio anddecreases methylation potential of cells and restores differentiationand/or developmental potential;

(b) modifying existing commercial or other media to enable knock down ofDNA methyltransferases 1, 3a, and/or 3b using interfering RNA technologyresulting in reduced levels of DNA methylation;

(c) modifying existing commercial or other media to enable knock down ofMTHFR and/or Cbs using interfering RNA technology, antisense, or otherknock down technologies resulting in decreased SAM:SAH ratio and DNAmethylation;

(d) modifying existing commercial or other media to increase thetranscription, protein expression and activity of GNMT resulting indecreased SAM:SAH ratio and DNA methylation;

(e) modifying existing commercial or other media to decrease SAM:SAHratio by altering levels of methyl donors and/or homocysteine and/orSAH.

Without wishing to be held to one explanation for the rationale behindthe present invention, it is believed that methyl groups provided fromthe diet, culture media, or the folate-dependent one-carbon pool areessential for normal function. Methyl group deficiency leads todown-regulation of transmethylation reactions. Methyl groups suppliedfrom the diet (or culture media), in the form of choline and methionineor from the folate-dependent one-carbon pool, must be activated to SAMto serve as substrates in numerous transmethylation reactions. BecauseSAH is a product of transmethylation reactions and a potent inhibitor ofmost SAM-dependent methyltransferases, the ratio of SAM/SAH is animportant index of transmethylation potential. Therefore, theintracellular supply of SAM is critical for transmethylation, but theregulation of this ratio is important as well.

The cytosolic enzyme GNMT functions to optimize transmethylationreactions by regulating the SAM/SAH ratio. When methyl groups areabundant and SAM levels elevated, GNMT disposes of the excess methylgroups by forming the essentially inactive metabolite sarcosine fromglycine. SAM also reduces the supply of methyl groups originating fromthe one-carbon pool by inhibiting MTHFR, the enzyme responsible for thesynthesis of 5-methyl-THF, the folate coenzyme that donates its methylgroup to homocysteine to form methionine. Because 5-methyl-THF alsobinds to GNMT and inhibits its activity, a decrease in 5-methyl-THFlevels due to inhibition of MTHFR by SAM results in an increase in theactivity of GNMT. Conversely, under conditions of decreased methylgroups and SAM, the inhibition of MTHFR is removed, leading to anincrease in 5-methyl-THF concentrations and subsequent inhibition ofGNMT. This ensures that methyl groups are conserved for importanttransmethylation reactions when methyl group availability iscompromised. Therefore, factors that inappropriately activate GNMT maylead to the down regulation of numerous methyltransferases that areimportant in the maintenance of an appropriate methylation state.

The present invention was driven by the need to accelerate developmentof new, more effective, efficient methods of cell reprogramming. Suchmethods impact the discovery and development of therapeutically valuablecells, including stem cells, new research tools for accelerating stemcell and other cell-based therapy research, propagation of valuable orendangered animals, and production of high value genetically engineeredanimals.

The invention is directed towards resolving problems with currentmethods for dedifferentiating cells, and redifferentiating them into newcells. Specifically, current methods, primarily based on SCNT, arehighly inefficient, and produce unreliable results. The new inventionavoids the use of nuclear transfer, and its associated limitations. SCNTunits exhibit defects in regulating DNA methylation, includingdeficiencies in global demethylation, which contributes to inefficientcell reprogramming. The present invention overcomes this specificproblem.

The present invention also will enable and improve the efficiency ofSCNT for the production of endangered animals, value added livestock andderivation of ES cells by improving the dedifferentiation (globaldemethylation) process required after reconstruction.

The invention also will enable development of new cell-basedtherapeutics that overcomes the critical problems of immune rejectionand limited source of material associated with the use of embryonic stemcells. By enabling the use of a patient's own somatic cells fordedifferentiation and redifferentiation into new cell types, theinvention will provide an immuno-compatible source of cells, from a widevariety of starting cell types.

Moreover, the field of stem cell research is highly limited at thecurrent time by inadequate cell cultures and media for maintaining cellsin dedifferentiated states, or for directing cells to differentiatealong specific lineages. The pathways, proteins, and related moleculesassociated with the new invention will enable development of newcultures and media that overcome these limitations. Similarly, thepresent invention will provide new markers of differentiation andredifferentiation, and the ability to produce new differentiated celllines that will speed drug discovery by providing and enhancing newapproaches for screening regenerative medicine drug candidates.

The field of cell-based therapeutics is highly limited at the currenttime due to limited sources of starting material and expensive methodsof isolation and expansion under GMP/GML guidelines. For example, in thecase of new cell therapy applications involving transplantation of isletcells to produce insulin in diabetic patients, current methods requiredat least two cadaver donors provide sufficient cells for just onepatient. The present invention is directed at overcoming theseshortcomings by significantly improving the amount of starting materialand desired product to be used. Moreover, such cells carry an inherentrisk of inducing adverse immune rejection in the recipient. The presentinvention is directed at overcoming this problem by offering theopportunity to use a patient's own cells as the source of startingmaterial.

The present invention is directed towards methods that can acceleratethe progress of reprogramming by regulating DNA methylation. Methodsaimed at altering the expression and/or activity of DNAmethyltransferases could have significant effects increasing theefficiency of reprogramming by altering the SAM:SAH ratio. One approachis to alter the regulation of methyl group metabolism by specificallyaltering the expression of key enzymes using compounds such as retinoicacid (RA) and retinoids. Administration of ATRA and other retinoidcompounds induces and activates GNMT, compromising SAM-dependenttransmethylation reactions, including the methylation of DNA, resultingin hypomethylation of genomic DNA. Others have used methylationinhibitors, such as 5-azacytidine and 5-aza-2′-deoxycytidine to decreaseDNA methylation levels and provide antitumor effects [82].

Another method is to overexpress a demethylase in somatic cells inculture. To date, an active demethylase has not been validated.

The key advantages of the present invention over prior methods include:

The invention does not involve the use of nuclear transfer, thus avoidsthe high level of inefficiency in nuclear reprogramming and actual celldevelopment.

The invention does not rely upon the use of embryonic stem cells asstarting materials for cell differentiation. Thus, it overcomes acritical limiting barrier in availability of starting material.

The invention can be used to improve embryonic and adult stem cellculture systems to minimize spontaneous differentiation.

The invention does not rely upon embryonic stem cells or other foreignmaterials for development of new regenerative cell therapies, and thusminimizes the potential for immune rejection by the patient.

The invention exploits natural metabolic processes by a simplenutritional approach, thus reducing the number of complicating factorsthat could contribute to failure, such as the cytotoxic affects inducedby compounds such as 5′-azacytidine and azacitadine (also known as Aza Cand manufactured under the trademark VIDAZA by Pharmion of Boulder,Colo.) or requiring a genetic alteration such as overexpressing anactive demethylase using a transgene system.

The invention can be used to improve SCNT.

The invention can be used to more efficiently improve the derivation ofES cells from SCNT-generated embryos (units).

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting relative levels of cytosine methylation inbovine serial nuclear transfer donor somatic cells after growth toconfluence in growth media alone (CON), growth media treated with DMSO(DMSO), and growth media treated with all-trans retinoic acid (ATRA).See Example 1.

FIG. 2 is a graph illustrating the level of GNMT gene expression (asmeasured by real time PCR) in bovine serial nuclear transfer donorsomatic cells after growth to confluence in growth media alone (CON),growth media treated with DMSO (DMSO), and growth media treated withall-trans retinoic acid (ATRA). See Example 1.

FIGS. 3A and 3B are photographs illustrating bovine cells treated asdescribed in Example 2. FIG. 3A shows cells after treatment with ATRA.FIG. 3B shows cells after positive staining with oil red-0 (a commonstain for lipids) following ATRA treatment. See Example 2.

FIGS. 4A and 4B are photographs showing murine 3T3 L1 cells treated withATRA followed by osteogenic induction media, as described in Example 4.FIG. 4A depicts cells that tested positive for osteoblast-like cellswith alkaline phosphatase staining. FIG. 4B depicts cells exhibitingcalcium phosphate mineralization with alizarin staining.

FIG. 5 is a photograph of a gel illustrating gene expression (by RT-PCR)for osteocalcin and cbfa1, osteoblast-specific markers, in murine 3T3 L1cells treated with ATRA followed by osteogenic induction media. SeeExample 4.

FIGS. 6A and 6B are photographs of gels illustrating the knock down ofthe Dnmt1 gene by siRNA, as described in Example 5. FIG. 6A depicts thespecific knock down of Dnmt1 using a Dnmt1 siRNA. FIG. 6B depicts thespecific knock down of lamin using a lamin siRNA.

FIG. 6C is a photograph of a gel showing that the knock down of Dnmt1 asshown in FIG. 6A persisted through at least the two-cell stage. SeeExample 5.

FIGS. 7A and 7B are graphs illustrating the effects of DNAmethyltransferase 3b siRNA transfection on murine NIH3T3 somatic cellmethylation, as described in Example 5.1. FIG. 7A depicts real time PCRresults showing a 79% decrease in Dnmt3b mRNA levels as compared tocontrols. FIG. 7B depicts HPLC results showing a 52% relative reductionin methylated cytosine in the cells.

ABBREVIATIONS AND DEFINITIONS

Unless otherwise described herein, the following definitions andabbreviations will apply throughout this disclosure:

ATRA means all-trans retinoic acid.

Cbs means cystathione-beta-synthase.

Cell Reprogramming means the process by which a terminallydifferentiated cell can be restored to a state where it has thepotential to differentiate into a new cell type.

Culture Medium or Growth Medium means a suitable medium capable ofsupporting growth of cells.

Demethylating Culture Medium or Demethylating Growth Medium means asuitable medium capable of supporting growth of cells in a manner thatinduces demethylation of DNA.

Differentiation means the process by which cells become structurally andfunctionally specialized during embryonic development.

DMSO means dimethyl sulfoxide.

DNA means deoxyribonucleic acid.

DNA Methylation means the attachment of a methyl group (a —CH3 group) toa cytosine (one of four nitrogenous bases found in DNA) in a eukaryoticDNA. This is done routinely, as a way to protect self DNA from theenzymes and chemicals produced to destroy foreign DNA, and as a way toregulate transcription of genes in the DNA.

DNMT means DNA methyltransferase.

Epigenetics refers to the state of DNA with respect to heritable changesin function without a change in the nucleotide sequence. Epigeneticchanges can be caused by modification of the DNA, such as by methylationand demethylation, without any change in the nucleotide sequence of theDNA.

FRET means fluorescence resonance energy transfer.

GAPDH means glyceraldehyde-3-phosphate dehydrogenase.

GNMT means glycine-N-Methyltransferase.

H3K4me means methylation of histone H3 at Lys4.

H3K9me2/3 means di/trimethylation of specific lysine residues such aslysine 9 on histone H3.

Histone means a class of protein molecules found in chromosomesresponsible for compacting DNA enough so that it will fit within anucleus. Prokaryotes do not have histones. However, the DNA of the genusThermoplasma (a cell-wall-less member of the domain Archaea) issurrounded by a highly basic DNA-binding protein which stronglyresembles the eukaryotic histones.

IBMX means isobutylmethylxanthine.

ICM means inner cell mass.

“Knock down” means to suppress the expression of a gene in agene-specific fashion. A cell that has one or more genes “knocked down,”is referred to as a knock-down organism or simply a “knock-down.”

MDS means myelodysplastic syndrome.

MTHFR means 5,10-methylenetetrahydrofolate reductase.

Oocyte means a developing female reproductive cell which divides bymeiosis into four haploid cells, forming one ovum that can be fertilizedby a sperm cell.

Pluripotent means capable of differentiating into cell types of the 3germ layers or primary tissue types.

RA means retinoic acid.

Reprogramming means removing epigenetic marks in the nucleus, followedby establishment of a different set of epigenetic marks. Duringdevelopment of multicellular organisms, different cells and tissuesacquire different programs of gene expression. These distinct geneexpression patterns appear to be substantially regulated by epigeneticmodifications such as DNA methylation, histone modifications and otherchromatin binding proteins [22, 23]. Thus each cell type within amulticellular organism has a unique epigenetic signature which isconventionally thought to become “fixed” and immutable once the cellsdifferentiate or exit the cell cycle. However, some cells undergo majorepigenetic “reprogramming” during normal development or certain diseasesituations.

RNA means ribonucleic acid.

RT-PCR means reverse transcribed polymerase chain reaction.

SAH means S-adenosylhomocysteine.

SAM means S-adenosylmethionine.

SCNT means somatic cell nuclear transfer.

siRNA means small interfering RNA, also known as short interfering RNAor silencing RNA.

Somatic cells mean any of the cells of an organism that havedifferentiated into the tissues, organs, etc. of the body (as opposed togerm cell—any of various cells, especially an egg or sperm cell, fromwhich a new organism can develop).

TE means trophoectoderm.

Totipotent means capable of developing into a complete embryo or organ.

TZD means Thiazolidinedione.

DETAILED DESCRIPTION OF THE INVENTION

The present invention involves inducing demethylation of a patient'sadult stem cells or fully differentiated somatic cells in a manner thatrestores differentiation potential and can be used as an autologoussource of therapeutic cells; or, using the demethylated cells to improvethe efficiency of SCNT and to more efficiently generate patient-specificES cells to be used for therapeutic purposes.

The present invention is specifically directed to specialized cell linesand a specialized cell culture. The methods for achieving both arecritical to the invention. In its basic sense, the methods involvedemethylation steps that yield reprogrammed cell lines. The factors thatenable demethylation are included in the cell culture medium, which isused to produce cells that can be differentiated into new cell types.

The present invention is directed to cell culture medium to which ATRAand/or siRNA can be added, or components adjusted sufficiently, toreduce DNA methylation levels to such an extent as to restoredifferentiation and/or developmental potential either by inducing withspecific differentiation factors such as, but not limited to cytokines,growth factors, transcription factors and/or using the demethylatedcells for SCNT.

The invention is directed to in vitro production of reprogrammed cellswhich have had differentiation potential (totipotential, pluripotential,multipotential) restored by demethylating the genome. The embodimentsinclude:

(a) treating with ATRA, siRNA, and/or similar or related molecules;

(b) knocking down DNA methyltransferases 1, 3a, and/or 3b usinginterfering RNA technology, antisense, or other mechanisms forinhibition;

(c) knocking down MTHFR and/or Cbs using interfering RNA technology;

(d) increasing the transcription, protein expression and activity ofGNMT;

(e) increasing levels of homocysteine in vitro;

(f) increasing levels of SAH;

(g) decreasing levels of SAM;

(h) decreasing levels of methyl donors such as, but not limited to,folate, methionine, choline, betaine, vitamins B6 and B12;

(i) using the cells to improve SCNT; and

(j) using the cells to more efficiently derive ES cells from SCNTreconstructed units (embryos).

Cells: For purposes of the present invention, the term “cell” or“cells,” unless specifically limited to the contrary, includes anysomatic cell, embryonic stem (ES) cell, adult stem cell, nucleartransfer (NT) units, and stem-like cells. A promising source of organsand tissues for transplantation lies in the development of stem celltechnology. Theoretically, stem cells can undergo self-renewing celldivision to give rise to phenotypically and genotypically identicaldaughters for an indefinite time and ultimately can differentiate intoat least one final cell type. By generating tissues or organs from apatient's own cells, transplant tissues can be generated to provide theadvantages associated with xenotransplantation without the associatedrisk of infection or tissue rejection.

Stem cells also provide promise for improving the results of genetherapy. A patient's own stem cells could be genetically altered invitro, then reintroduced in vivo to produce a desired gene product.These genetically altered stem cells would have the potential to beinduced to differentiate to form a multitude of cell types forimplantation at specific sites in the body, or for systemic application.Alternately, heterologous stem cells could be genetically altered toexpress the recipient's major histocompatibility complex (MHC) antigen,or no MHC, to allow transplant of those cells from donor to recipientwithout the associated risk of rejection.

Stem cells are defined as cells that have extensive and indefiniteproliferation potential that differentiate into several cell lineages,and that can repopulate tissues upon transplantation. The quintessentialstem cell is the embryonal stem (ES) cell, as it has unlimitedself-renewal and pluripotent differentiation potential. These cells arederived from the inner cell mass of the blastocyst, or can be derivedfrom the primordial germ cells from a post-implantation embryo(embryonal germ cells or EG cells). ES and EG cells have been derivedfrom mouse, and more recently also from non-human primates and humans.When introduced into mouse blastocysts or blastocysts of other animals,ES cells can contribute to all tissues of the mouse (animal). Whentransplanted in post-natal animals, ES and EG cells generate teratoma,which again demonstrates their pluripotency. ES and EG cells can beidentified by positive staining with the antibodies SSEA1 and SSEA4.

At the molecular level, ES and EG cells express a number oftranscription factors highly specific for these undifferentiated cells.These include Oct-4 and Rex-1. Also found are the LIF-R and thetranscription factors Sox-2 and Rox-1, even though the latter two arealso expressed in non-ES cells. Oct-4 is a transcription factorexpressed in the pregastrulation embryo, early cleavage stage embryo,cells of the inner cell mass of the blastocyst, and in embryoniccarcinoma (EC) cells. Oct-4 is down-regulated when cells are induced todifferentiate in vitro and in the adult animal oct-4 is only found ingerm cells. Several studies have shown that Oct-4 is required formaintaining the undifferentiated phenotype of ES cells, and plays amajor role in determining early steps in embryogenesis anddifferentiation. Oct-4, in combination with Rox-1, causestranscriptional activation of the Zn-finger protein Rex-1, and is alsorequired for maintaining ES in an undifferentiated state. Likewise,Sox-2 is needed together with Oct-4 to retain the undifferentiated stateof ES/EC cells and to maintain murine (but not human) ES cells. Human ormurine primordial germ cells require presence of LIF. Another hallmarkof ES cells is presence of telomerase, which provides these cells withan unlimited self-renewal potential in vitro.

Stem cells have been identified in several organ tissues. The bestcharacterized is the hematopoietic stem cell. This is a mesoderm-derivedcell that has been purified based on cell surface markers and functionalcharacteristics. The hematopoietic stem cell, isolated from bone marrow,blood, cord blood, fetal liver and yolk sac, is the progenitor cell thatreinitiates hematopoiesis for the life of a recipient and generatesmultiple hematopoietic lineages [93-100]. When transplanted intolethally irradiated animals or humans, hematopoietic stem cells canrepopulate the erythroid, neutrophil-macrophage, megakaryocyte andlymphoid hemopoietic cell pool. In vitro, hemopoietic stem cells can beinduced to undergo at least some self-renewing cell divisions and can beinduced to differentiate into the same lineages as is seen in vivo.Therefore, this cell fulfills the criteria of a stem cell. Stem cellswhich differentiate only to form cells of hematopoietic lineage,however, are unable to provide a source of cells for repair of otherdamaged tissues, for example, heart or lung tissue damaged by high-dosechemotherapeutic agents.

A second stem cell that has been studied extensively is the neural stemcell [101-103]. Neural stem cells were initially identified in thesubventricular zone and the olfactory bulb of fetal brain. Untilrecently, it was believed that the adult brain no longer contained cellswith stem cell potential. However, several studies in rodents, and morerecently also non-human primates and humans, have shown that stem cellscontinue to be present in adult brain. These stem cells can proliferatein vivo and continuously regenerate at least some neuronal cells invivo. When cultured ex vivo, neural stem cells can be induced toproliferate, as well as to differentiate into different types of neuronsand glial cells. When transplanted into the brain, neural stem cells canengraft and generate neural cells and glial cells. Therefore, this celltoo fulfills the definition of a stem cell.

A third tissue specific cell that has been named a stem cell is themesenchymal stem cell (MSC), initially described by Fridenshtein [104].Mesenchymal stem cells, originally derived from the embryonal mesodermand isolated from adult bone marrow, can differentiate to form muscle,bone, cartilage, fat, marrow stroma, and tendon. During embryogenesis,the mesoderm develops into limb-bud mesoderm, tissue that generatesbone, cartilage, fat, skeletal muscle and possibly endothelium. Mesodermalso differentiates to visceral mesoderm, which can give rise to cardiacmuscle, smooth muscle, or blood islands consisting of endothelium andhematopoietic progenitor cells. Primitive mesodermal or mesenchymal stemcells, therefore, could provide a source for a number of cell and tissuetypes. A number of mesenchymal stem cells have been isolated [105-117].Of the many mesenchymal stem cells that have been described, all havedemonstrated limited differentiation to form only those differentiatedcells generally considered to be of mesenchymal origin. To date, themost multipotent mesenchymal stem cell reported is the cell isolated byPittenger, et al [118], which expresses theSH2.sup.+SH4.sup.+CD29.sup.+CD44.sup.+CD71.sup.+CD90.sup.+CD106.sup.+CD120a.sup.+CD124.sup.+CD14.sup.−CD34.sup.−CD45.sup.−phenotype.This cell is capable of differentiating to form a number of cell typesof mesenchymal origin, but is apparently limited in differentiationpotential to cells of the mesenchymal lineage, as the team who isolatedit noted that hematopoietic cells were never identified in the expandedcultures.

Other stem cells have been identified, including gastrointestinal stemcells, epidermal stem cells, and hepatic stem cells, also termed ovalcells [119-121]. Most of these are less well characterized.

Compared with ES cells, tissue specific stem cells have lessself-renewal ability and, although they differentiate into multiplelineages, they are not pluripotent. No studies have addressed whethertissue specific cells express markers described above of ES cells. Inaddition, the degree of telomerase activity in tissue specific stemcells has not been fully explored, in part because large numbers ofhighly enriched populations of these cells are difficult to obtain.

Until recently, it was thought that organ specific stem cells could onlydifferentiate into cells of the same tissue. A number of recentpublications have suggested that adult organ specific stem cells may becapable of differentiating into cells of different tissues. A number ofstudies have shown that cells transplanted at the time of a bone marrowtransplant can differentiate into skeletal muscle [122-123]. This couldbe considered within the realm of possible differentiation potential ofmesenchymal cells that are present in marrow. Jackson published thatmuscle satellite cells can differentiate into hemopoietic cells, again aswitch in phenotype within the splanchnic mesoderm [124]. Other studieshave shown that stem cells from one embryonal layer (for instancesplanchnic mesoderm) can differentiate into tissues thought to bederived during embryogenesis from a different embryonal layer. Forinstance, endothelial cells or their precursors detected in humans oranimals that underwent marrow transplantation are at least in partderived from the marrow donor [125-126]. Thus, visceral mesoderm and notsplanchnic mesoderm, such as MSC, derived progeny are transferred withthe infused marrow. Even more surprising are the reports demonstratingboth in rodents and humans that hepatic epithelial cells and biliaryduct epithelial cells are derived from the donor marrow [127-129].Likewise, three groups have shown that neural stem cells candifferentiate into hemopoietic cells. Finally, Clarke et al. reportedthat neural stem cells injected into blastocysts can contribute to alltissues of the chimeric mouse [130].

Transplantation of tissues and organs generated from heterologousembryonic stem cells requires either that the cells be furthergenetically modified to inhibit expression of certain cell surfacemarkers, or that the use of chemotherapeutic immune suppressors continuein order to protect against transplant rejection. Thus, althoughembryonic stem cell research provides a promising alternative solutionto the problem of a limited supply of organs for transplantation, theproblems and risks associated with the need for immunosuppression tosustain transplantation of heterologous cells or tissue would remain. Anestimated 20 immunologically different lines of embryonic stem cellswould need to be established in order to provide immunocompatible cellsfor therapies directed to the majority of the population [132].

Using cells from the developed individual, rather than an embryo, as asource of autologous or allogeneic stem cells would overcome the problemof tissue incompatibility associated with the use of transplantedembryonic stem cells, as well as solve the ethical dilemma associatedwith embryonic stem cell research. The greatest disadvantage associatedwith the use of autologous stem cells for tissue transplant thus farlies in their limited differentiation potential. A number of stem cellshave been isolated from fully-developed organisms, particularly humans,but these cells, although reported to be multipotent, have demonstratedlimited potential to differentiate into multiple cell types.

Even though stem cells with multiple differentiation potential have beenisolated previously by others, a demethylated cell with the potential todifferentiate into a wide variety of cell types of different lineages,including fibroblasts, osteoblasts, chondrocytes, adipocytes, skeletalmuscle, endothelium, stroma, smooth muscle, cardiac muscle, nueral cellsand hemopoietic cells, has not been described. If cell and tissuetransplant and gene therapy are to provide the therapeutic advancesexpected, a demethylated cell with restored or expanded differentiationand/or developmental potential is needed, either as a source for patientspecific therapeutic cells, or as a more efficient donor cell for SCNT,or as an object for screening of differentiation factors or compoundsthat might be used to assist in inducing differentiation along specificlineages.

Cell Culture Media: The present invention utilizes cell culture media,growth factors, and methods for inducing DNA demethylation of cells andgrowing and maintaining cultures of demethylated cells. The mediaprovides for the growth and maintenance of cells and can be used toscreen for additional factors and useful combinations of factors. Theability to grow cells in a substantially undifferentiated state usingthe cell culture media, growth factors, and methods provided hereinprovides important benefits including the ability to produce cell lineshaving multiple genetic modifications (as in the application of genetherapy) with important therapeutic applications.

The cell culture media may include a growth medium that is effective tosupport the growth of demethylated cells; a nutrient serum effective tosupport the growth of demethylated cells; non-essential and essentialamino acids, and a pyruvate salt. Optionally, the cell culture media mayalso include a reducing agent.

Another non-limiting example of a suitable culture medium useful inpracticing the present invention includes a variety of growth mediaprepared with a base of Dulbecco's minimal essential media (DMEM). TheDMEM can be supplemented with a variety of supplements including fetalcalf serum, glutamine and/or sodium pyruvate. Preferably the DMEMincludes 15% fetal calf serum, 2 mM glutamine and 1 mM sodium pyruvate.

Another example of a suitable culture medium is glucose- andphosphate-free modified human tubal fluid media (HTF) supplemented with15% fetal calf serum, 0.2 mM glutamine, 0.5 mM taurine, and 0.01 mM eachof the following amino acids: asparagine, glycine, glutamic acid,cysteine, lysine, proline, serine, histidine, and aspartic acid.

Growth Factor: The growth medium can include any serum or serum-basedsolution that supplies nutrients effective to maintain the growth andviability of demethylated cells. Examples of such serum include, withoutlimitation, fetal bovine serum (FBS) and fetal calf serum (FCS). Forexample, the FBS may be provided in a concentration of between about 1%and about 25%. In particular, the FBS may be provided in a concentrationof between about 2.5% and about 20%. In one embodiment, demethylatedcells are grown in 10% FBS.

A growth factor may also be provided to assist in the derivation andmaintenance of cultures of demethylated cells in a substantiallyundifferentiated state. The identities and effective concentrations ofsuch growth factors can be determined using the methods as describedherein or using techniques known to those of skill in the art ofculturing cells. For example, one or more of the following factors canbe used at the stated final concentration: forskolin([3R-(3α,4αβ,5B,6B,6aα,10α,10αβ,10α)]-5-(acetyloxy)-3-ethenyldodecahydro-6,10,10b-trihydroxy-3,4-a,7,7,10a-pentamethyl-1H-naphtho[2,1-b]pyran-1-one)at 10 μM, cholera toxin at 10 μM, isobutylmethylxanthine (IBMX) at 0.1mM, dibutyrladenosine cyclic monophosphate (dbcAMP) at 1 mM. In anotherembodiment, the growth factor is basic fibroblast growth factor (bFGF),more specifically, human recombinant basic fibroblast growth factor(bFGF), in the range of about 1-10 ng/ml.

Another factor is growth media harvested from cultures of humanembryonal carcinoma (EC) cells. In a particular example, human NTERA-2EC cells (ATCC accession number CRL 1973) are grown to confluence inDMEM supplemented with 10% fetal calf serum or mouse ES cells are grownto confluence in DMEM supplemented with 15% fetal calf serum, 2 mMglutamine, 1000 U/ml LIF. Growth media is harvested daily over severaldays, passed through a 0.22 micron filter and frozen at −80° C. Thishuman EC or mouse ES “conditioned” media is added to the demethylatedgrowth media in empirically determined amounts, as judged by the effecton demethylated cell growth and viability.

In another embodiment the growth media includes ligands for receptorsthat activate the signal transduction gp130, either by binding to areceptor that associates with gp 130 or by binding directly to andactivating gp 130. For example, human recombinant leukemia inhibitoryfactor (LIF) at about 1000 U/ml to 2000 U/ml or oncostatin-M at 10 U/ml,can be used.

Tissue Culture Antibiotics: Typically, embryonal germ (EG) medium alsocontains commonly used tissue culture antibiotics, such as penicillinand streptomycin. An effective amount of factors are then added daily toeither of these base solutions to prepare human EG growth media of theinstant invention. The term “effective amount” means the amount of suchdescribed factor as to permit a beneficial effect on human EG growth andviability of human EG cells using judgment common to those of skill inthe art of cell culturing and by the teachings supplied herein.

Typically, the demethylating culture medium also contains commonly usedtissue culture antibiotics, such as penicillin and streptomycin. Aneffective amount of these factors are then added daily to either ofthese base solutions to prepare demethylating growth media of theinstant invention. The term “effective amount” as used herein is theamount of such described factor as to permit a beneficial effect ondemethylation and viability of cells using judgment common to those ofskill in the art of cell culturing and by the teachings supplied herein.

Ranges: Table 1 provides ranges for the ingredients useful in the cellculture medium of the present invention.

TABLE 1 Medium Most preferable Preferable Acceptable Working pH range7.0-7.4 7.0-7.4 7.0-7.4 Component mg/L mg/L mg/L CaCl (anhyd.) 200.0050-350 1-500 Fe(NO₃)3—9H₂O 0.10 0.01-0.2  0.001-1.0   KCl 400.00100-600   1-1000 MgSO₄ (anhyd.) 97.67 10-250 1-500 NaCl 6400.005000-8000  1000-10000  NaHCO₃ 3700.00 2000-6000  500-10000 NaH₂PO₄—H₂O125.00 50-250 1-500 D-Glucose 1000.00 1000-5000   1-10000 Phenol Red15.00 15.00 15.00 Sodium Pyruvate 110.00  10-200—  1-1000 L-Arginine-HCl84.00 50-150 10-500  L-Glutamine 584.00 300-600  100-900  Glycine 30.0010-60  1-100 L-Histadine-HCl—H₂O 42.00 10-100  1-1000 L-Isoleucine105.00 10-250 1-500 L-Leucine 105.00 10-250 1-500 L-Lysine-HCl 146.0010-250 1-500 L-Methionine 30.00 10-100 1-500 L-Phenylalanine 66.0010-100 1-500 L-Serine 42.00 10-100 1-500 L-Threonine 95.00 50-250 1-500L-Tryptophan 16.00 5-50 1-100 L-Tyrosine-2Na—2H₂O 104.00 10-500 1-900L-Valine 94.00 50-150 10-500  D-Ca pantothenate 4.00 1-10 0.1-50  Chlorine Chloride 4.00 1-10 0.1-50   Folic Acid 4.00 1-10 0.1-50  i-Inositol 7.20 1-10 0.1-50   Niacinamide 4.00 1-10 0.1-50  Pyridoxal-HCl 4.00 1-10 0.1-50   Riboflavin 0.40 1-10 0.1-50  Thiamine-HCl 4.00 1-10 0.1-50  

An example of a cell culture medium which can be used in the presentinvention is alpha-MEM (Chemicon International, Temecula Calif.) asdescribed in the following Table 2:

TABLE 2 Working pH range 7.0-7.4 Component mg/L Inorganic Salts CaCl₂(anhyd.) — KCl 400.00 KH₂PO₄ — MgCl₂ (anhyd.) 94.70 MgSO₄ (anhyd.) —NaCl 6500.00 NaHCO₃ 2000.00 NaH₂PO₄—H₂O 1154.00 NaH₂PO₄ (anhyd.) — OtherComponents D-Glucose 2000.00 HEPES — Lipoic Acid — Phenol Red 10.00Sodium Pyruvate — Amino Acids L-Alanine — L-Arginine-HCl 126.00L-Asparagine-H₂O — L-Aspartic Acid — L-Cystine-2HCl 32.40L-Cystine-HCl—H₂O — L-Glutamic Acid — L-Glutamine 292.00 Glycine —L-Histidine 31.00 L-Histidine-HCl—H₂O 42.00 L-Isoleucine 52.00 L-Leucine52.00 L-Lysine 58.00 L-Lysine-HCl — L-Methionine 15.00 L-Phenylalanine32.00 L-Proline — L-Serine — L-Threonine 48.00 L-Tryptophan 10.00L-Tyrosine-2Na—2H₂O 52.00 L-Valine 46.00 Vitamins L-Ascorbic Acid —Biotin — D-Ca pantothenate 1.00 Chlorine Chloride 1.00 Folic Acid 1.00i-Inositol 2.00 Niacinamide 1.00 Pyridoxal-HCl 1.00 Riboflavin 0.10Thiamine-HCl 1.00 Vitamin B12 — Deoxyribonucleosides 2′ Deoxyadenosine —2′ Deoxyxytidine-HCl — 2′ Deoxyguanosine — Thymidine — RibonucleosidesAdenosine — Cytidine — Guanosine — Uridine —

General Techniques for Culturing

General methods in molecular genetics and genetic engineering aredescribed in the current editions of Sambrook et al. [84], Miller & Calmeds. [85], and F. M. Ausubel et al. eds. [86]. Cell biology, proteinchemistry, and antibody techniques can be found in J. E. Colligan et al.eds. [87], J. S. Bonifacino et al., [88], and J. E. Colligan et al. eds.[89]. Reagents, cloning vectors, and kits for genetic manipulationreferred to in this disclosure are available from commercial vendorssuch as BioRad (Hercules, Calif.), Stratagene (La Jolla, Calif.),Invitrogen Corporation (Carlsbad, Calif.), ClonTech (Mountain View,Calif.), and Sigma-Aldrich Co (St. Louis, Mo.).

Cell culture methods are described generally in Freshney ed, [90], M. A.Harrison & I. F. Rae [91], and K. Turksen ed. [92]. Cell culturesupplies and reagents are available from commercial vendors such asGibco/BRL (Gaithersburg, Md.), Nalgene-Nunc International (Rochester,N.Y.), Sigma Chemical Co. (St. Louis, Mo.), Hyclone (Logan, Utah),Chemicon International (Temecula, Calif.) and ICN Biomedicals (CostaMesa, Calif.).

The demethylated cells can be grown on the plate in addition to thefeeder cells. Alternatively, the feeder cells can be first grown toconfluence and then mitotically inactivated (e.g., by irradiation) toprevent further growth. Alternatively the demethylated cells can begrown without feeder layer cells. Such an approach has the advantage ofsimplifying the management of the cell culture as the growth of only oneset of cells, the demethylated cells, need only be monitored.

Culturing the Established Demethylated Cells: Once established, thedemethylated cells can be cultured under the above-described conditionedmedium using a variety of techniques. In one example, a container holdsfeeder cells in a non-conditioned medium. A matrix of lysed feeder cellsis prepared using standard methods. The demethylated cells to becultured are then added atop the matrix along with the conditionedmedium. Alternatively, the demethylated cells can be grown on livingfeeder cells using methods known in the art. The growth of thedemethylated cells is then monitored to determine the degree to whichthe cultured cells have become differentiated. A marker for alkalinephosphatase can be used to ascertain which cells have differentiated.When a sufficient number of cells have differentiated, or when theculture has grown to confluence, at least a portion of theundifferentiated cells can be passaged. The determination to passage thecells and the techniques for accomplishing such passaging can beperformed using standard techniques well known in the art.

Dedifferentiating Somatic Cells in Culture: Cells are grown incustom-designed growth media deficient in one or more methyl donors orcoenzymes (folate, betaine, choline, Vitamin B6, Vitamin B12 andmethionine), or containing increased amounts of homocysteine or SAH.Formulations are based on Chemicon International Specialty Media(Phillipsburg, N.J.) or American Type Culture Collection(ATCC)-recommended media.

The formula deficiencies or supplements are shown in Table 3 as follows:

TABLE 3 Medium Most Preferable Preferable Acceptable [M] Folate 0 0-2.26× 10⁻⁵ 0-4.53 × 10⁻⁴ Betaine 0 0-8.53 × 10⁻⁵ 0-1.71 × 10⁻³ Choline 00-3.38 × 10⁻⁵ 0-6.76 × 10⁻⁴ VitaminB₆ 0 0-8.10 × 10⁻⁹ 0-1.62 × 10⁻⁷VitaminB₁₂ 0 0-1.23 × 10⁻⁹   2.46 × 10⁻⁸ Methionine 0 0-3.35 × 10⁻⁴  6.67 × 10⁻³ Homocysteine 0.0005 0.0001-0.001 0.00001-0.002 SAH 0.00050.0001-0.001 0.00001-0.002

As an example, the culture medium can be supplemented with 10% fetalbovine serum, 1% penicillin-streptomycin and 0.1% fungizone. Dialyzedfetal bovine serum (Invitrogen) is added to the folate deficient mediumto eliminate folic acid in the serum. Cells are maintained at 37° C. in95% humidity and 5% CO₂. Media is changed every 3-4. days, and cells arepassaged at ˜90% confluency repeatedly for up to 6 weeks. Endpointsinclude reduced cytosine methylation levels, reduced SAM:SAH ratios, andincreased GNMT expression, activity and protein abundance.

To inhibit Dnmt activity and cytosine methylation, cells are grown inthe following media:

(a) media treated with Dnmt1, 2, 3a, and/or 3b siRNA (Dharmacon, Inc.);

(b) media treated with RG108 (Analytical Systems Laboratory, LSU Schoolof Veterinary Medicine);

(c) media treated with 5-AzadCyd (Sigma).

A brief description of siRNA transfection according to manufacturer'sprotocol is provided below.

To stimulate GNMT synthesis and activity, cells are grown in mediatreated with ATRA (Sigma). To protect ATRA from light exposure, theprocedures are performed in subdued lighting and treatment flasks arewrapped in foil.

To inhibit histone deacetylation and induce demethylation, cells aregrown in media treated with TSA and/or VPA (Sigma). Treatmentconcentrations are listed, but are not limited to, the conditionsillustrated in Table 4 as follows:

TABLE 4 Medium Most Preferable Preferable Acceptable [μM] Dnmt siRNA^(a)0.05 0.01-0.1  0.001-0.2   RG108^(b) 20  10-100   1-200 AzadCyd^(c) 0.250.1-0.5 0.01-1.0  ATRA^(d) 0.05 0.01-0.1  0.01-200 TSA^(e) 0.30 0.1-3.00.01-300 VPA^(f) 20 × 10³ 1-40 × 10³ 0.1-200 × 10³ ^(a)Manufacturerrecommendations of [100 nM] for 75% gene knockdown; 20-40% CpG islanddemethylation with [40 nM] Dnmt1 and/or 3b siRNA [133]; 79% geneknockdown and ~50% global genomic demethylation with [200 nM] Dnmt3bsiRNA in NuPotential's preliminary studies. ^(b)20% demethylation with[100 μM] RG108 [134]. ^(c)40% demethylation with [0.5 μM] AzadCyd [134].^(d)~25% demethylation with [100 nM] ATRA in our preliminary studies.^(e)5% demethylation with [0.2 μM] TSA [135]. ^(f)4.8% demethylationwith [20 mM] VPA [135].

Cells are maintained at 37° C. in 95% humidity and 5% CO₂. Media ischanged every 3-4 days with fresh treatment, and cells are passaged at˜90% confluency repeatedly for up to 6 weeks; see Dnmt siRNAtransfection methods below for exception. Cell viability/cytotoxicityand growth rates are determined by relative cell counts. Endpointsinclude reduced cytosine methylation levels, reduced SAM:SAH ratios,increased GNMT expression, activity and protein abundance, and decreasedDnmt expression.

Dnmt siRNA transfection: At approximately 70-80% confluency, cells aretransfected in growth media containing DharmaFECT (Dharmacon, Inc.)transfection reagent, Dnmt1, 2, 3a, and/or 3b siRNA (Dharmacon, Inc.),cyclophillin b siRNA (positive control siRNA), or drosophilanon-targeting siRNA (negative control siRNA); Dnmt2 has not beendetermined to be functional and may serve as an additional control.Transfection is carried out according to manufacturer's protocol. Cellsare incubated in transfection media for 48 hours. As per manufacturer'srecommendation, if cell toxicity is observed after 24 hours,transfection media is replaced with growth media and incubation iscontinued for an additional 24 hours; wells exhibiting 80% viabilityafter 48 hour incubation are used for differentiation.

Alternative Embodiments

The invention also is directed to development of a new assay based onuse of reporter genes such as beta galactamase, luciferase, betaglucosidase, or others that provide the capability of monitoring theimpact of GNMT expression and/or activity on demethylation andrestoration of cell differentiation potential.

The invention is further directed to development of a novel method ofSCNT using dedifferentiated donor cells.

The invention also is directed to development of a novel method toderive embryonic stem cells from SCNT reconstructed units (embryos).

EXAMPLES

The following Examples are included solely to aid in a more completeunderstanding of the subject invention. The Examples do not limit thescope of the invention described herein in any fashion.

Example 1 Effects of RA on Bovine Somatic Cell Methylation

Bovine serial nuclear transfer donor somatic cells (a cell line that hasnever produced a successful live animal and which is characterized bylow blastocyst development rates (<10%, P<0.001) and low pregnancyinitiation rates (<1%, P<0.05)) were cultured in T-75 flasks untilconfluent in growth media (CON), or growth media treated with DMSO, orATRA (DMSO vehicle); 10 nM, 50 nM, and 100 nM ATRA concentrations wereused. Following AMA treatment, the cells were collected and DNA from thecells was isolated. The DNA was then completely digested into singlenucleotides for reverse-phase HPLC. Reverse-phase HPLC was performed asdescribed by Cezar et al. [76] to measure the relative levels ofmethylated cytosine residues.

As illustrated in FIG. 1, HPLC revealed a 25% reduction in relativemethylated cytosine in those cells cultured in 100 nM ATRA-treated mediacompared to CON. Those treated with DMSO or 10-50 nM ATRA exhibited an8-10% reduction in methylated cytosine compared to CON. These resultsindicate that ATRA can reduce methylation status, in vitro, even incells resistant to reprogramming, such as those obtained through serialnuclear transfer.

As illustrated in FIG. 2, real-time RT-PCR analysis demonstratedupregulation of the gene encoding glycine-N-methyltransferase (GNMT),the key enzyme regulating the SAM:SAH ratio and cellular methylationpotential. (The results are depicted as the ratio of GNMT toglyceraldehyde-3-phosphate dehydrogenase (GAPD). The GAPD expressionlevel is used to normalize the expression of the GNMT to a knownmarker.) Note that the upregulation of GNMT as shown in FIG. 2 happenssimultaneously with the decreased DNA methylation shown in FIG. 1,Moreover, RT-PCR confirmed upregulation and strong expression of Oct4,the most diagnostic mammalian stem cell/pluripotency marker, as well asSox2 and nanog (data not shown). The RT-PCR experiments were performedusing a commercially available kit and following the manufacturer'sinstructions (TAQMAN-brand RT-PCR assays, Applied Biosystems, FosterCity, Calif.) Others have demonstrated that demethylation of Oct4 is arequirement for efficient reprogramming of mice somatic nuclei [20].

These data indicate that exposure to ATRA increases expression of GNMTand likely activity of the GNMT enzyme, resulting in globaldemethylation (including demethylation of the Oct4 promoter andsubsequent expression). Decreased DNA methylation is consistent withdedifferentiation, while increased expression of Oct4 is consistent withrestoration of differentiation potential. To our knowledge, this is thefirst demonstration of a stem cell marker being induced in a mammaliansomatic cell by altering single-carbon metabolism. The results aresignificant because they show that a mammalian cell can be induced toexpress a marker normally found only in undifferentiated stem cells,thus indicating that the cell can be reprogrammed to differentiate intoa distinct cell type.

Example 2 Effects of RA and Adipogenic Induction Cocktail on BovineSomatic Cells

To determine whether differentiation potential was restored, bovinesomatic cells were cultured in T-75 flasks until confluent in CON, DMSO,or ATRA media (same RA as in Example 1). Following ATRA treatment, cellswere collected for RNA isolation or transferred to 6-well plates andcultured in an adipogenic induction cocktail that included DMEM, FBS,insulin, IBMX and dexamethasone. The IBMX was removed and TZD was added48 hours later. Cells remained in the induction media for 11-12 days,then were either oil red-O-stained (Aldrich, Milwaukee, Wis.) toascertain the presence of lipid droplets, or collected for RNAisolation. RNA was reverse-transcribed to cDNA for RT-PCR for stem cellmarkers (Oct 4, Sox2, Nanog, AP) and adipogenic markers (PPARγ, LPL,FAS), or used directly in one-step quantitative real-time RT-PCR forGNMT. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as anormalizing/housekeeping gene.

As illustrated in FIG. 3A, during exposure to ATRA, significantmorphological changes were observed, including development of structuresthat were remarkably similar to embryoid bodies. As illustrated in FIG.3B by the arrow, positive oil red-O staining was also observed afterinduction, indicating the presence of lipid. Gene expression analyses byPCR revealed induction of PPARγ, a known marker of adipocytedifferentiation (data not shown). These results are significant becausethey suggest that somatic cells can be induced to re-differentiate intoother cell types.

Example 3 Effects of RA and Adipogenic Induction Cocktail on PrimaryHepatocytes

Primary rat hepatocytes were grown in media treated with 50-200 nM ATRA.Following treatment, cells were cultured under the same comparativeconditions as described above for the bovine somatic cells, except anadipogenic induction cocktail (as described in EXAMPLE 2) was added tothe T-75 flasks. Morphological changes were observed during ATRAtreatment, and within 48 hours after initial exposure to inductionmedia, these changes became pronounced. By day 4 of exposure toadipogenic induction media, these morphological changes were accompaniedby the beginnings of spheroid body development and distinctproliferation of cells as evidenced by confluency, a state not reachedin control cells. These observations suggest that these primaryhepatocytes that are terminally differentiated re-entered the cell cyclefollowing ATRA treatment and exposure to adipogenic induction media inculture and proliferated significantly following the treatments.

Example 4 Effects of RA and Osteogenic Induction Cocktail on MurineSomatic Cells

3T3 L1 cells were grown in media treated with 50-200 nM ATRA. FollowingATRA treatment, cells were transferred to 6-well plates and exposed toosteogenic induction media containing DMEM, FBS, ascorbic acid2-phosphate, dexamethasone, beta glycerophosphate, and vitamin D for 21days. As illustrated in FIG. 4A and FIG. 4B, following osteogenicinduction, positive staining for osteoblast-like cells (FIG. 4A) andcalcium phosphate mineralization (FIG. 4B) was observed with alkalinephosphatase and alizarin staining, respectively. As illustrated in FIG.5, RT-PCR revealed induction of osteocalcin and cbfa1, known markers ofosteoblast-specific differentiation.

Example 5 Production of Cell Lines and Production of Cell Cultures thatProduce Cells with Restored Differentiation Potential by Knocking DownDNA Methyltransferases 1, 3A, and/or 3B using Interfering RNA Technology

Knocking down DNA methyltransferases 1, 3a, and/or 3b using interferingRNA technology results in reduced levels of DNA methylation. Bymodifying the culture media to incorporate siRNA technology, selectivegene expression suppression can be accomplished. The methodology forachieving this impact-including foundation media, specific treatmentsand modification to that media, and methods of confirming knock downpotential is described below.

Altering single-carbon metabolism and/or inhibiting DNMT activityinduces global DNA demethylation which can restore differentiationpotential. De-differentiated cell lines capable of re-differentiatinginto multiple lineages can be produced by global epigeneticreprogramming. Preliminary studies designed to analyze the impact ofDNMT gene knockdown using siRNA technology on global DNA demethylationin a bovine donor cell line for SCNT and a murine fibroblast cell linedemonstrate how this is accomplished.

Example 5.1 Effects of DNA Methyltransferase 1 siRNA Transfection onBovine Somatic Donor Cell Methylation and Developmental Potential

Bovine oocytes aspirated from abattoir ovaries were matured overnight inmaturation medium (medium 199; Biowhittaker) supplemented withluteinizing hormone (10 IU/ml; Sigma), estradiol (1 mg/ml; Sigma), andFBS (10%; tlyclone, Logan, Utah) at 38.5° C. in a humidified 5% CO₂incubator. Bovine nuclear transfer was performed as described byForsberg et al. [131]. A human siRNA to Dnmt1 used by Robert et al.[140]) was ordered from Dharmacon RNA Technologies (Lafayette, Colo.).The sequence was as follows: AAGCAUGAGCACCGUUCUCC.dT.dT. (SEQ. ID.NO: 1) This was duplexed and desalted as per manufacturer'sinstructions. Transfection of siRNA occurred according to LIPOFECTINmanufacturer protocol Briefly, 2 μM siRNA in 1× Universal buffer wasmixed with LIPOFECTIN (Invitrogen) transfection reagent with serum freegrowth medium (Opti-MEM; Invitrogen). (Invitrogen) and Robert et al[140]. Prior to transfection, culture medium was removed from cells thatwere ˜40-60% confluent and 3 ml transfection reagent solution was added,with final transfection concentrations of 200 nM siRNA and 0.6%LIPOFECTIN transfection reagent.

The cells were incubated for 24 hours at 37° C. and washed withAmnioMAX-brand media containing serum (Invitrogen). Cells weretransfected twice prior to analysis. Control and Test samples weregenerated simultaneously and include:

1. Cells treated with media alone.

2. Cells treated with transfection reagent (LIPOFECTIN) alone.

3. Cells treated with control siRNA (Lamin A/C; Dharmacon).

4. Cells treated with Test siRNA (Dnmt1; Dharmacon)

The following figures and legends summarize these studies. FIG. 6A andFIG. 6B illustrate the knockdown of Dnmt1 by siRNA. Dnmt1 wasefficiently knocked down after siRNA treatment of bovine donor cells forSCNT (FIG. 6A, Lane D) while actin expression was not affected (FIG. 6A,Lanes G-I). In contrast, exposure to lamin siRNA knocked down laminexpression, (FIG. 6B, lane C) but not Dnmt1 (FIG. 6A, lane C). HPLCanalysis confirmed an approximately 40% decrease in global methylationlevels in cells treated with Dnmt 1 siRNA, and the cells weresubsequently used for SCNT. FIG. 6C illustrates that knockdown of Dnmt1persisted through at least the 2 cell stage (lanes C-F) when compared tocontrols (lanes A & B), and blastocyst development rates weresignificantly higher when compared to controls (50% vs 21%, p<0.05).

Example 5.2 Effects of DNA Methyltransferase 3b siRNA Transfection onMurine Somatic Cell Methylation

NIH3T3 cells were transfected with 200 nM murine Dnmt3b siRNA (DharmaconRNA Technologies, Lafayette, Colo.). Pooled siRNA sequences are asfollows: GCAAUGAUCUCUCUAACGU (SEQ. ID. NO: 2); GGAAUGCGCUGGGUACAGU (SEQ.ID. NO: 3); UAAUCUGGCUACCUUCAAU (SEQ. ID. NO: 4); GCAAAGGUUUAUAUGAGGG(SEQ. ID. NO: 5). The transfection protocol was optimized in our labessentially as described by the manufacturer. Briefly, 2 μM siRNA in1×siRNA buffer was mixed with DharmaFECT transfection reagent with serumfree growth medium (DMEM; Hyclone, Logan, Utah). Prior to transfection,culture medium was removed from cells that were ˜70-80% confluent andtransfection reagent solution was added to cover the surface. Finaltransfection concentrations were 200 nM siRNA and 0.6% DharmaFECTtransfection reagent in a 2 ml total volume per well of E-well plates.

Cells were incubated at 37° C. in 5% CO₂ for 24-48 hours. Allexperiments included the following Control and Test samples intriplicate:

1. Untreated cells (media alone)

2. Mock transfection (no siRNA; DhannaFECT only)

3. Positive control siRNA (cyclophillin b)

4. Negative control siRNA (drosophila non-targeting)

5. Test siRNA (Dnmt 3b)

Dnmt3b mRNA expression was determined by real-time RT-PCR (using aTAQMAN-brand RT-PCR kit and following the manufacturer's instructions).The results are shown in FIG. 7A; n=2. Methylation levels weredetermined via HPLC. The results are shown in FIG. 7B; n=3.

As illustrated in FIG. 7A, real-time RT-PCR revealed a 79% decrease inDnmt3b mRNA levels relative to cyclophillin b in cells transfected withDnmt3b siRNA as compared to control cells. As illustrated in FIG. 7B,HPLC revealed a 52% reduction in methylated cytosine in cellstransfected with Dnmt3b siRNA as compared to control cells. These dataindicate that Dnmt3b gene knockdown can reduce methylation status, invitro.

Example 6 New Media for DNA Methyltransferase siRNA Transfection inHuman and Murine Somatic Cells

Additional human and murine Dnmt siRNAs have been purchased fromDharmacon (Lafayette, Colo.). Pooled siRNA sequences are as follows:

1. Human Dnmt1: GGAAGAAGAGUUACUAUAA; (SEQ. ID. NO: 6)GAGCGGAGGUGUCCCAAUA; (SEQ. ID. NO: 7) GGACGACCCUGACCUCAAA (SEQ. ID. NO:8) GAACGGUGCUCAUGCUUAC. (SEQ. ID. NO: 9) 2. Human Dnmt3a:GCACAAGGGUACCUACGGG; (SEQ. ID. NO: 10) CAAGAGAGCGGCUGGUGUA; (SEQ. ID.NO: 11) GCACUGAAAUGGAAAGGGU; (SEQ. ID. NO: 12) GAACUGCUUUCUGGAGUGU.(SEQ. ID. NO: 13) 3. Human Dnmt3b: GAAAGUACGUCGCUUCUGA; (SEQ. ID. NO:14) ACAAAUGGCUUCAGAUGUU; (SEQ. ID. NO: 15) GCUCUUACCUUACCAUCGA; (SEQ.ID. NO: 16) UUUACCACCUGCUGAAUUA. (SEQ. ID. NO: 17) 4. Murine Dnmt1: 1-GGAAAGAGAUGGCUUAACA; (SEQ. ID. NO: 18) GCUGGGAGAUGGCGUCAUA; (SEQ. ID.NO: 19) GAUAAGAAACGCAGAGUUG; (SEQ. ID. NO: 20) GGUAGAGAGUUACGACGAA.(SEQ. ID. NO: 21) 5. Murine Dnmt3a: CGCGAUUUCUUGAGUCUAA; (SEQ. ID. NO:22) CGAAUUGUGUCUUGGUGGA; (SEQ. ID. NO: 23) AAACAUCGAGGACAUUUGU; (SEQ.ID. NO: 24) CAAGGGACUUUAUGAGGGU. (SEQ. ID. NO: 25)

All protocols will be optimized in our lab essentially as described bythe manufacturer. Briefly, 2 μM siRNA in 1×siRNA buffer is mixed withDharmaFECT transfection reagent with serum free growth medium (DMEM;Hyclone, Logan, Utah). Prior to transfection, culture medium will beremoved from cells that are ˜70-80% confluent and transfection reagentsolution will be added to cover the surface. Final transfectionconcentrations are 200 nM siRNA and 0.6% DharmaFECT transfection reagentin a 2 ml total volume per well of 6-well plates.

Cells will be incubated at 37° C. in 5% CO₂ for 24-48 hours. Allexperiments will include the following samples in triplicate: untreatedcells, mock transfection (no siRNA, DharmaFECT only), positive controlsiRNA (cyclophillin b), negative control siRNA (drosophilanon-targeting), and test siRNA.

Real time RT-PCR will be used to confirm gene knockdown. HPLC will beused to measure relative cytosine methylation levels.

Example 7 New Media that Knocks Down MTHFR and/or Cbs using InterferingRNA Technology

Knocking down MTHFR and/or Cbs using interfering RNA technology resultsin decreased SAM:SAH ratio and DNA methylation. Appropriate siRNAs arepurchased commercially (Dharmacon RNA technologies, Lafayette, Colo.)and are duplexed and desalted according to manufacturer's instructions.Pooled human and murine siRNA sequences are as follows:

Human MTHFR: AGUGAGAGCUCCAAAGAUA; (SEQ. ID. NO: 26) GAAGUGAGUUUGGUGACUA;(SEQ. ID. NO: 27) GACCAAAGAGUUACAUCUA; (SEQ. ID. NO: 28)GCAAGUGUCUUUGAAGUCU. (SEQ. ID. NO: 29) Human Cbs: AGACGGAGCAGACAACCUA;(SEQ. ID. NO: 30) CACCACCGCUGAUGAGAUC; (SEQ. ID. NO: 31)GGACGGUGGUGGACAAGUG; (SEQ. ID. NO: 32) GGAAGAAGUUCGGCCUGAA. (SEQ. ID.NO: 33) Murine MTHFR: CGCCAUGGCUACAGAGUAA; (SEQ. ID. NO: 34)GCGGAAACCAGCCUGAUGA; (SEQ. ID. NO: 35) CAGAAGGCCUACCUCGAAU; (SEQ. ID.NO: 36) CAUACGAGCUGCGGGUCAA. (SEQ. ID. NO: 37) Murine Cbs:GCAAACAGCCUAUGAGGUG; (SEQ. ID. NO: 38) GCAAAGUCCUCUACAAGCA; (SEQ. ID.NO: 39) GAUCGAAGAUGCUGAGCGA; (SEQ. ID. NO: 40) CAACCCUUUGGCACACUA. (SEQ.ID. NO: 41)

Positive and negative siRNAs are purchased from Dharmacon and useddirectly in transfections.

All protocols are optimized essentially as described by themanufacturer. Briefly, 2 μM siRNA in 1×siRNA buffer is mixed withDharmaFECT transfection reagent with serum free growth medium (DMEM;Hyclone, Logan, Utah). Prior to transfection, culture medium is removedfrom cells that are ±70-80% confluent and transfection reagent solutionis added to cover the surface. Final transfection concentrations are 200nM siRNA and 0.6% DharmaFECT transfection reagent in a 2 ml total volumeper well of 6-well plates.

Cells are incubated at 37° C. in 5% CO2 for 24-48 hours. All experimentswill include the following samples in triplicate: untreated cells, mocktransfection (no siRNA, DharmaFECT only), positive control siRNA(cyclophillin b), negative control siRNA (drosophila non-targeting), andtest siRNA.

Real time RT-PCR is used to confirm gene knockdown. HPLC is used tomeasure relative cytosine methylation levels.

Example 8 New Media that Increases the Transcription, Protein Expressionand Activity of GNMT

Increasing the transcription, protein expression and activity of GNMTresults in decreased SAM:SAH ratio and DNA methylation. This involvesidentification of compounds, extracts, molecules that have similareffects as ATRA such as increasing GNMT activity, and/or decreasingSAM:SAH ratio.

Depleting methyl donors in vitro results in decreased SAM:SAH ratio andDNA methylation. The medias are custom designed and deficient in one ormore methyl donors or coenzymes (folate, betaine, choline, Vitamin B6,Vitamin B12 and methionine and/or others); or, have increased amounts ofhomocysteine; or combinations of deficient methyl donors/coenzymesand/or increased levels of homocysteine. The formulations are based onHyClone's 1640 medium (HyClone, Logan, Utah) with modifications as shownin Table 5:

TABLE 5 Experimental Medium Control Medium Component: g/L g/L Medium 1:Betaine 0 0.03 Medium 2: Choline 0 0.03 Medium 3: Vitamin B₆ 0 0.000005Medium 4: Vitamin B₁₂ 0 0.000005 Medium 5: Methionine 0 0.15 Medium 6:Folate 0 0.03

Example 9 Media that Increase Levels of Homocysteine In Vitro

Increasing levels of homocysteine in vitro results in decreased SAM:SAHratio and DNA methylation. The medias are custom designed to containincreased amounts of homocysteine; or combinations of deficient methyldonors/coenzymes and/or increased levels of homocysteine. Theformulations are based on HyClone's 1640 medium with modifications asshown in Table 6:

TABLE 6 Experimental Medium Control Medium Component: g/L g/L Medium 6:Homocysteine 0.0625 0 Medium 7: Folate 0 0.03 Homocysteine 0.0625 0Medium 8: Betaine 0 0.03 Homocysteine 0.0625 0 Medium 9: Choline 0 0.03Homocysteine 0.0625 0 Medium 10: Vitamin B₆ 0 0.03 Homocysteine 0.0625 0Medium 11: Vitamin B₁₂ 0 0.03 Homocysteine 0.0625 0

Culturing cells in defined media having reduced or depleted levels of aspecific methyl donor, or multiple methyl donors, or containingincreased levels of homocysteine, or increased levels of homocysteine incombination with reduced or depleted levels of a specific methyl donor,or methyl donors, results in decreased SAM:SAH ratio and DNAmethylation.

Example 10 High Throughput Assay using GNMT Promoter

Factors that increase expression of, and/or activate GNMT, are likely todown regulate DNA methyltransferases and reduce global methylationlevels. A β-lactamase-based assay (and/or other assays described below)are developed to identify inducers (and suppressors) of GNMT expressionby induction of GNMT transcription.

β-lactamase assay (Invitrogen) is used to develop cell based assays thatcan be used to identify drugs, molecules and extracts that induce DNAdemethylation through the single-carbon metabolism pathway and toidentify drugs, molecules and extracts that induce redifferentiation ofin vitro restored pluri- and multipotent cell lines. The initialobjective is to develop a cell based assay that can be used to screenfor drugs, molecules and extracts that induce expression of GNMT.

The basis of this assay will be the GENEBLAZER technology (Invitrogen),a gene reporter system based on a membrane permeant fluorescenceresonance energy transfer (FRET)-based substrate (CCF2-AM) that providesviable cell staining, compatibility with fluorescence-activated cellsorting, ratiometric read-out and an optimized platform for highthroughput screening. Excitation of CCF2 at 405 nm results in ablue/green emission due to the absence of FRET, respectively, in theCCF2 substrate molecule. The cell permeable CCF2-AM consists of 2fluorophores, 7-hydroxycoumarin and fluorescein, linked by acephalosporin core.

Excitation of coumarin at 405 nm results in FRET to the fluoresceinmoiety, which emits green fluorescence light at 530 nm. β-lactamasecleaves the β-lactam moiety within the cephalosporin core of the CCF2-AMsubstrate molecule into two separate fluorophores, resulting in FRETdisruption.

Excitation of the cells at 405 nm results in blue fluorescence emissionby coumarin, detected at 460 nm. The presence of β-lactamase in thecells causes a change from green to blue fluorescent cells. Thus, duringFACS analysis cells with different emission characteristics can becollected.

A 1.8 kb DNA fragment that contains the 5′ upstream region of human GNMTis PCR amplified from genomic DNA. The primers used are:

Pr4564: 5′-GGGGTACCAGCATCTT-3′ (SEQ. ID. NO: 42) and

Pr6391: 5′-GCGAGATCTCCTGCGCCGCGCCTGGCT-3′ (SEQ. ID. NO: 43).

PCR conditions are as follows: 1.5 mM MgCl₂, 200 nm primers, 35 cyclesconsisting of an annealing step at 60° C. for 1 min, extension at 72° C.for 2 min and 35 cycles.

After amplification, SDS and EDTA are added to 0.1% and 5 mM,respectively. DNA is precipitated with 2.5 M ammonium acetate and 70%ethanol. The fragment is digested with KpnI and BglII and the fragmentisolated by elution from agarose after gel electrophoresis. The fragmentis ligated into vector p7-blaM carrying the β-lactamase M codingsequence (blaM) gene. The construct is transfected into CHO-K1 cellsusing Lipofectamine 2000 brand reagent (Invitrogen) as transfectionreagent. Vector without insert is transfected as a negative control.Stable cells are selected by resistance to Zeocin. Cells are grown inphenol-red free MEM-α medium with 10% charcoal-treated serum, pen/strepand 10 mM HEPES buffer.

Clones with low β-lactamase expression are selected by FACS analysis.Cells are trypsinized and washed twice with FACS buffer[phosphate-buffered saline (PBS), 5% charcoal treated FBS] and loaded ata concentration of 1×10⁶ cells/ml with appropriate GENEBLAZERtechnology, cell permeable FRET-based substrate (CCFs-AM) in the darkwith mild shaking for 1 hour.

Cells are harvested by centrifugation and resuspended in FACS buffer atthe same cell concentration. Cells are sorted using a Becton-DickensonFACStarplus cell sorter (Franklin Lakes, N.J.). An argon UV laser isused to excite cells loaded with substrate. Clones with responding greenfluoroescene are collected into 96-well plates containing growth mediawith 200 g/ml Zeocin and grown to a final cell population of 6×106.Stable cell lines are tested for inducibility by treatment with ATRA.

Prior experiments have demonstrated a dose response of GNMTtranscription to ATRA. This response is used as criteria for asuccessful assay. Specifically, an assay is considered successful when a3 fold induction is detected by treatment with 100 nM ATRA and confirmedby real-time PCR. Clones with an ATRA inducible β-lactamase expressionare identified and used for further development.

It is also within the scope of the present invention to utilize assayswith similar applications using other reporter genes such asluceriferase, beta-galactosidase, or others.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims following the Bibliography.

BIBLIOGRAPHY

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1. A method for reprogramming a eukaryotic cell, the method comprising:(a) decreasing S-adenosylmethione-to-S-adenosylhomocysteine ratio(SAM-to-SAH ratio) in the eukaryotic cell.
 2. The method of claim 1,further comprising: (b) reducing levels of 5-methylcytosine in DNAwithin the eukaryotic cell.
 3. The method of claim 2, wherein steps (a)and (b) are performed simultaneously.
 4. The method of claim 2, whereinstep (b) comprises specifically suppressing expression, activity, orexpression and activity of DNA methyltransferases within the eukaryoticcell.
 5. The method of claim 4, wherein step (b) comprises contactingthe cell with a DNA methyltransferase inhibitor.
 6. The method of claim4, wherein step (b) comprises contacting the cell with asuppression-effective amount of a small-interfering ribonucleic acid(siRNA) dimensioned and configured to suppress expression of DNAmethyltransferases.
 7. The method of claim 1, wherein step (a) comprisescontacting the cell with a siRNA selected from the group consisting ofSEQ. ID. NOS: 1-41.
 8. The method of claim 1, wherein step (a) comprisesincreasing expression, activity, or both expression and activity ofglycine-N-methyl transferase within the eukaryotic cell.
 9. The methodof claim 8, wherein step (a) comprises contacting the cell with anamount of a retinoic acid effective to increase expression, activity, orboth expression and activity of glycine-N-methyl transferase within thecell.
 10. The method of claim 1, wherein the SAM-to-SAH ratio isdecreased to 0.1 or less.
 11. The method of claim 1, wherein theSAM-to-SAH ratio is decreased to 0.5 or less.
 12. The method of claim 1,wherein the SAM-to-SAH ratio is decreased to 1.00 or less.
 13. A methodfor reprogramming a eukaryotic cell, the method comprising: (a)specifically suppressing expression, activity, or expression andactivity of DNA methyltransferases within a eukaryotic cell; andsimultaneously (b) increasing expression, activity, or both expressionand activity of glycine-N-methyl transferase within the eukaryotic cell.14. The method of claim 13, wherein step (a) comprises contacting thecell with a suppression-effective amount of a siRNA dimensioned andconfigured to suppress expression of DNA methyltransferases.
 15. Themethod of claim 13, wherein step (a) comprises contacting the cell witha suppression-effective amount of a siRNA dimensioned and configured tosuppress to reduce 5-methylated cytosines in DNA in the cell by at leastabout 5%.
 16. The method of claim 13, wherein step (b) comprisescontacting the cell with an amount of a retinoic acid effective toincrease expression, activity, or both expression and activity ofglycine-N-methyl transferase within the cell.
 17. The method of claim16, wherein step (b) comprises contacting the cell with all-transretinoic acid.
 18. The method of claim 16, wherein step (b) comprisescontacting the cell with a compound that binds a retinoic acid receptor.19. The method of claim 13, further comprising, simultaneously withsteps (a) and (b): (c) specifically suppressing within the cellexpression, activity, or expression and activity of an enzyme selectedfrom the group consisting of 5,10-methylenetetrahydrofolate reductaseand cystathione-beta-synthase.
 20. The method of claim 13 comprisingcontacting the cell with a siRNA selected from the group consisting ofSEQ. ID. NOS: 1-41.
 21. A method to change differentiation potential ina eukaryotic cell, the method comprising contacting the eukaryotic cellin vitro with an amount of a siRNA, wherein the siRNA is dimensioned andconfigured to suppress expression of DNA methyltransferasesspecifically, provided that the siRNA does not suppress expression ofglycine-N-methyl transferase, and wherein the amount is effective toinduce genome-wide DNA demethylation, wherein cellular differentiationpotential is increased.
 22. The method of claim 21, wherein theeukaryotic cell is selected from the group consisting of stem cells,progenitor cells, somatic cells, and cells subjected to somatic cellnuclear transfer (NT cells).
 23. The method of claim 21, comprisingcontacting the cell with a siRNA that is dimensioned and configured tosuppress specifically the expression of a DNA methyltransferase selectedfrom the group consisting of DNA methyltransferase 1 (Dnmt 1), DNAmethyltransferase 3a (Dnmt 3a), and DNA methyltransferase 3b (Dnmt 3b).24. The method of claim 21, further comprising contacting the cell withan amount of a compound effective to increase expression of, activityof, or both expression of and activity of, glycine-N-methyl transferase.25. The method of claim 24, comprising contacting the cell with retinoicacid.
 26. The method of claim 24, comprising contacting the cell withall-trans retinoic acid.
 27. The method of claim 24, comprisingcontacting the cell with a composition comprising, in combination, thesiRNA and all-trans retinoic acid.
 28. A method to increasedifferentiation potential in a differentiated cell, the methodcomprising contacting a differentiated cell, in vitro, with acomposition comprising, in combination: (i) a knockdown-effective amountof a small-interfering ribonucleic acid (siRNA), wherein the siRNA isdimensioned and configured to suppress expression of DNAmethyltransferases specifically; (ii) a knockdown-effective amount of asiRNA dimensioned and configured to suppress expression of an enzymeselected from the group consisting of 5, 10-methylenetetrahydrofolatereductase and cystathione-beta-synthase; and (iii) a compound effectiveto increase expression of, activity of, or both expression of andactivity of, glycine-N-methyl transferase, wherein the cell is contactedwith the composition for a time effective to induce genome-wide DNAdemethylation within the cell, whereby cellular differentiationpotential is increased.
 29. The method of claim 28, comprisingcontacting the cell with a composition comprising a siRNA that isdimensioned and configured to suppress specifically the expression of aDNA methyltransferase selected from the group consisting of DNAmethyltransferase 1 (Dnmt 1), DNA methyltransferase 3a (Dnmt 3a), andDNA methyltransferase 3b (Dnmt 3b).
 30. The method of claim 28, whereinthe compound effective to increase expression of, activity of, or bothexpression of and activity of, glycine-N-methyl transferase, is retinoicacid.
 31. The method of claim 28, wherein the compound effective toincrease expression of, activity of, or both expression of and activityof, glycine-N-methyl transferase, is all-trans retinoic acid.
 32. Themethod of claim 28, comprising contacting the cell with a siRNA selectedfrom the group consisting of SEQ. ID. NOS: 1-41.
 33. A method fordetermining developmental potential of a cell, the method comprisingdetermining SAM-to-SAH ratio in the cell, wherein a SAM-to-SAH ratio ofless than about 1.0 indicates increased developmental potential of thecell.
 34. The method of claim 33, further comprising measuringmethylation of cytosine residues present in the DNA of the cell, whereinmethylation less than about 10% indicates increased developmentalpotential of the cell.
 35. A method for controlling differentiationpotential in a cell, the method comprising; (a) maintaining SAM-to-SAHratio in the cell to less than about 1.0; and (b) maintainingmethylation of cytosine residues in DNA of the cell to less than about40%.
 36. The method of claim 34, comprising: (a) maintaining theSAM-to-SAH ratio in the cell to less than about 0.5; and (b) maintainingthe methylation of cytosine residues in DNA of the cell to less thanabout 20%.
 37. A cell culture medium for increasing differentiationpotential in a differentiated cell disposed within the culture mediumthe culture medium comprising a base medium sufficient to maintainviability of the cell, in combination with a factor that reducesactivity of methyl donors present in the cell culture medium.
 38. Thecell culture medium of claim 37 wherein the factor is selected from thegroup consisting of homocysteine and all-trans retinoic acid.
 39. Thecell culture medium of claim 37 wherein the factor is asuppression-effective amount of a siRNA dimensioned and configured tosuppress expression of DNA methyltransferases.
 40. A method foridentifying compounds that affect cell differentiation, the methodcomprising: (a) reducing percentage of cytosines in DNA of a cell thatare methylated by at least about 5%, wherein the cell has a firstphenotype prior to the reduction; (b) reducing SAM-to-SAH ratio in thecell to less than about 1.0; and then (c) contacting the cell with acompound suspected of affecting cell differentiation; (d) comparing thefirst phenotype of the cell to phenotype of the cell after step (c),wherein appearance after step (c) of a phenotype different from thefirst phenotype indicates that the compound has an affect on celldifferentiation.
 41. The method of claim 40, wherein step (a) comprisesreducing the percentage of cytosines in the DNA of the cell that aremethylated by at least about 5%.
 42. The method of claim 40, whereinstep (a) comprises reducing the percentage of cytosines in the DNA ofthe cell that are methylated by at least about 10%.
 43. The method ofclaim 40, wherein the cell includes a reporter system to monitor changesin transcription.
 44. The method of claim 43, wherein the reportersystem measures presence of an enzyme activity selected from the groupconsisting of luciferase activity, beta-lactamase activity, andbeta-galactosidase activity.